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WO2006106166A1 - Souche vive attenuee de staphylococcus aureus servant de vaccin contre la mammite bovine - Google Patents

Souche vive attenuee de staphylococcus aureus servant de vaccin contre la mammite bovine Download PDF

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Publication number
WO2006106166A1
WO2006106166A1 PCT/ES2006/000164 ES2006000164W WO2006106166A1 WO 2006106166 A1 WO2006106166 A1 WO 2006106166A1 ES 2006000164 W ES2006000164 W ES 2006000164W WO 2006106166 A1 WO2006106166 A1 WO 2006106166A1
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Prior art keywords
staphylococcus aureus
strain
live attenuated
aureus
deletion
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PCT/ES2006/000164
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English (en)
Spanish (es)
Inventor
Ricardo De La Fuente Lopez
Gustavo Dominguez Bernal
Jose Antonio Orden Gutierrez
Susana MARTINEZ PULGARÍN
Jose antonio RUÍZ SANTA-QUITERIA
Dolores Cid Vazquez
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Universidad Complutense de Madrid
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Universidad Complutense de Madrid
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0004—Oxidoreductases (1.)
    • C12N9/0065—Oxidoreductases (1.) acting on hydrogen peroxide as acceptor (1.11)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/02—Bacterial antigens
    • A61K39/085—Staphylococcus
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/04—Antibacterial agents
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/20—Bacteria; Culture media therefor
    • C12N1/205—Bacterial isolates
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/36—Adaptation or attenuation of cells
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y111/00—Oxidoreductases acting on a peroxide as acceptor (1.11)
    • C12Y111/01—Peroxidases (1.11.1)
    • C12Y111/01006—Catalase (1.11.1.6)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/52—Bacterial cells; Fungal cells; Protozoal cells
    • A61K2039/522—Bacterial cells; Fungal cells; Protozoal cells avirulent or attenuated
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
    • C12R2001/44—Staphylococcus
    • C12R2001/445—Staphylococcus aureus

Definitions

  • the present invention falls within the field of Animal Health. More specifically, the invention refers to the production of a live strain of
  • Staphylococcus aureus attenuated by means of the deletion of two genetically stable genes, without antibiotic resistance markers and with the ability to proliferate and persist in mammary epithelial cells. It constitutes a new vaccine strain for ruminant mastitis.
  • Staphylococcus aureus is a very ubiquitous bacterium, a natural inhabitant of the skin and mucous membranes of mammals and the cause of various diseases in humans and domestic animals. In the latter, S. aureus is mainly involved in intramammary infections, which cause very important economic losses in bovine, sheep and goat production.
  • the ⁇ -toxin or ⁇ -hemolysin is a sphingomyelinase C dependent on Mg 2+ that degrades the sphingomyelin present in the outer layer of phospholipids of the cell membrane.
  • the cell lysis that it produces correlates with the sphingomyelin content of the membrane, therefore, ruminant erythrocytes, which have the highest percentage of sphingomyelin of all mammals, are the most sensitive.
  • Most strains of S. aureus isolated from bovine intramammary infections 75-100%) produce this hemolysin (encoded by the hlb gene), while human strains rarely express it (Aarestrup et al. 1999. APMIS.
  • S. aureus produces numerous extracellular enzymes such as hyaluronidase, nuclease, lipase, catalase, phosphatase, staphylokinase and proteases that have also been related to the pathogenicity of the bacterium in intramammary infections (Sutra and Poutrel. J Med Microbiol. 1994. 40: 79 -89), although, at the moment, the possible mode of pathogenic action of these enzymes is unknown.
  • Catalase is an enzyme that breaks down hydrogen peroxide generated during cellular metabolism in water and molecular oxygen. It has often been suggested that bacterial catalases may play a relevant role in the protection of microorganisms against bactericidal action derived from the oxidative metabolism of phagocytic cells. Thus, in various microorganisms, such as Nocardia asteroides (Beaman et al. 1985. Infect Immun. 47: 135-141), Mycobacterium tuberculosis (Manca et al. 1999. Infect Immun. 67: 74-79), Candida albicans (Wysong et al. 1998. Infect Immun. 66: 1953-1961; Nakagawa et al. 2003.
  • aureus catalase positive by measuring the survival capacity of the bacteria in the liver of inoculated roots through an intravenous route and concluded that the absence of caylase activity did not decrease the virulence of S. aureus.
  • the gene that encodes caialase (katA) in S. aureus has been sequenced and characterized (Sanz e ⁇ al. 2000. Microbiology. 146: 465-475).
  • S. aureus Although S. aureus has been considered as an extracellular pathogen, several studies in vivo and in vitro have shown that it is capable of adhering and being internalized by a wide variety of cell ips, being able to survive and, sometimes, multiply in the interior of epicelial cells (Almeida e al. 1996. J. Dairy Sci. 79: 1021-1026; Bayles et al. 1998. Infect Immun. 66: 336-342; Kahl et al. 2000. Infeci Immun. 68: 5385-5392; Brouille ⁇ e ⁇ al. 2003. Microb Pa ⁇ hog. 35: 159-168; Hess e ⁇ al. 2003. J Surg Res.
  • the invention consists in the construction of a live attenuated strain, of S. aureus, to be used as a vaccine, incapable of producing catalase and ⁇ -toxin activity, genetically stable and without antibiotic resistance markers.
  • Live strain is considered to be that strain that maintains its ability to invade and multiply inside its host, but without developing the pathogenic picture.
  • the method used to carry out this invention meets the essential requirements of biological safety for new generation vaccines. On the one hand, it ensures the stability of the mutation since the reversion to the wild phenotype is totally impeded due to the absence of targets required for homologous or illegitimate recombination from natural populations of bacteria. On the other hand, the constructed strain lacks antibiotic resistance markers.
  • the double mutation has a clear effect increasing the proliferation and persistence of the mutant in the MAC-T cell line of mammary epithelial cells and the persistence in the cell line of macrophages J774A.1, observing a synergistic and potentiating effect between both mutations (see Figures 4 and 5).
  • the proliferation of the mu ⁇ aeia is more len ⁇ a than the one of the parental strain, reaching the maximum of viable n ⁇ racellular bacterae more far.
  • the greater permissiveness for the replication of muierie in mammary epielial cells is a consequence of its lower toxicity for this cell type.
  • one aspect of this invention includes a vaccination kit for mammals against infectious diseases caused by S. aureus, which comprises the elements and adjuvants necessary to contain and vehicular the vaccine strain.
  • Another application of this invention consists in the use of the attenuated strain as an ideal candidate to be used as a vaccine vector for the expression or transport of heterologous antigens favored by its greater persistence in cells of the immune system together with its lower toxicity and its greater persistence in the inside mammary epithelial cells with respect to the wild strains of S. aureus.
  • FIG. 1 Shows the scheme of the construction of the thermosensitive plasmid pERhlb.
  • the oligonucleotides used in this study for the amplification of DNA fragments were designed from sequences of the genome of S. aureus COL (TIGR Microbial Datábase).
  • the numbers 1, 2, 3 and 4 correspond to the nomenclature of the oligonucleotides described in SEQ ID NO: 1, 2, 3 and 4, respectively.
  • FIG. 1 Scheme of the construction of the thermosensitive plasmid pERkat.
  • the numbers 5, 6, 7 and 8 correspond to the nomenclature of the oligonucleotides described in SEQ ID NO: 5, 6, 7 and 8, respectively.
  • FIG. 4 Intracellular survival of S. aureus 2386, its double katA / hlb mutant (S. aureus CECT 7061) and said mutanie supplemented with the plasmid containing both genes, in the macrophage cell line J774A.1 for 24 hours.
  • strain 2386 With a rhombus, strain 2386 is represented, with a square the double mutant and a double triangle mediated the complementary double muanie. In the ordinates axis, the percentage (%) of viable intracellular bacteria was repressed. This damage corresponds to the mean number of viable bacteria, plus-minus its standard deviation ( ⁇ SD), expressed in percentage referred to the total of bacteria present in the intracellular environment at time zero.
  • the time (T) in hours (h) is indicated on the abscissa axis.
  • S. aureus 2386 was used as a wild strain to conserie the Muiah strains
  • an analysis of variance was used.
  • p was less than 0.05 (statistically significant differences)
  • the Tukey-Kramer test was performed for a 95% confidence level.
  • Example 1 Construction of a mutant by deletion of the hlb gene, a mutant by deletion of the katA gene and a double mutant by deletion of both katA / hlb genes of S. aureus.
  • two fragments located in front and behind, respectively, of the hlb gene were amplified by PCR (see Figure 1).
  • the fragments obtained were joined by recombinant PCR following the technique described by Vallejo et al. (1994) and were cloned in pE194t, a plasmid derived from S.
  • Hlb deletion mutants were selected on Columbia agar with 5% lamb blood because they lacked b-hemolysis halo and were CAMP negative in the presence of Rhodococcus equi (Brzin, et al. 1990. Monbl Bakteriol. 273 (2 ): 179-83). Finally, the deletion of the hlb gene was confirmed by PCR techniques.
  • the katA deletion mutant was constructed in the same manner described above. For this, the pairs of oligonucleotides described in SEQ ID NO: 5 and 6 on the one hand and SEQ ID NO: 7 and 8 on the other were used to amplify the fragments located in front of and behind the katA gene, respectively (see figure 2). With the resulting plasmid, pERkat, the wild strain S. aureus 2386 was transformed obtaining mutants by deletion of katA. The katA deletion mutants were selected in solid medium due to the absence of catalase activity when they were contacted with 3% hydrogen peroxide. Likewise, the deletion of the katA gene was checked by PCR.
  • the double mutant by deletion of hlb / katA was constructed by transforming with the plasmid pERkat the mutant by deletion of hlb already constructed and following the same es ⁇ raiegia described for the mutant by deletion of katA.
  • This double muierie is deposited and registered in the Spanish Type Culinary Collection (CECT), University of Valencia, Burjasso ⁇ Campus, Research Building, 46100 Burjasso ⁇ (Valencia), with reference CECT 7061.
  • Example 2 Intracellular survival assays in J774A.1 and MAC-T cell lines. To study whether the loss of caylase activity, the lack of bioxine production or the absence of the two acivities affected survival and in vitro proliferation of S. aureus, intracellular survival studies were conducted in two different cell lines: macrophages J774A.1 and MAC-T mammary epithelial cells. The macrophages constitute one of the essential pieces of innate immunity, and participate actively in the presence of anigens to induce a satisfactory acquired immunity. Mammary epiyelia cells were used as an in vitro study model of the behavior of S. aureus in the mammary gland of ruminants.
  • the J774A.1 and MAC-T cell lines were handled with Dulbecco's modified Eagle medium (DMEM) to which 10% fetal bovine serum (FBS) and 1% Pen / S ⁇ rep / Fungizones Mix (Biowhi ⁇ ker) were added and incubations were done at 37 0 C with 5% CO2.
  • DMEM Dulbecco's modified Eagle medium
  • FBS fetal bovine serum
  • Pen / S ⁇ rep / Fungizones Mix Biowhi ⁇ ker
  • aureus 2386 its muiá ⁇ es by deletion of katA, hlb and katA / hlb, as well as the complemen ⁇ ados mutan ⁇ es ⁇ res, were cultivated during 18-20 hours in medium BHI to 37 0 C with agi ⁇ a
  • the cul ⁇ ivos were centrifuged, washed twice with iampon phosphaio saline (PBS) pH 7, were resuspended in PBS + 20% Glycerol and stored at -8O 0 C in 1 ml aliquots.
  • the bacterial concentration of the aliquots was determined after freezing by sowing serial dilutions in BHI plates.
  • Example 3 Calculation of lethal doses 50 (LD 50 ) in mice.
  • the lethal dose fifty (LD 50 ) was estimated after inoculating mice intraperitoneally, as detailed below.
  • mice 4-week-old Swiss female mice (21-23 g) were used, which were arranged in 5 groups of 5 animals each and to which water and food were supplied ad libitum under normal conditions of light and temperature. The animals were inoculated intraperitoneally with 0.4 ml of bacterial suspensions and deaths occurred every 24 hours for 7-10 days.
  • the DL 50 were calculated by the method of Reed and Muench (1938. Am J Hyg. 27: 493-497).
  • the DL 50 of the mutant by deletion of katA was lower than that of the wild strain S. aureus 2386, although in the analysis of variance these differences were not esistically significant.
  • the Calaslasa by itself is not a factor that significantly implies virulence.

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Abstract

Souche vive atténuée de Staphylococcus aureus inapte à produire une activité catalase y ß-toxine par combinaison des déletions. La formation du double mutant s'effectue par déletion en phase de tous les gènes codant la catalase, katA et la ß-toxine, hbl, par l'intermédiaire de la double recombinaison homologue. La souche est non virulente, génétiquement stabale et sans marqueurs de résistance antibiotique, elle se prête à une utilisation comme vaccin contre la mammite.
PCT/ES2006/000164 2005-04-07 2006-04-06 Souche vive attenuee de staphylococcus aureus servant de vaccin contre la mammite bovine Ceased WO2006106166A1 (fr)

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ESP200500809 2005-04-07
ES200500809A ES2307351B2 (es) 2005-04-07 2005-04-07 Cepa viva atenuada de staphylococcus aureus como vacuna en mastitis de rumiantes.

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US9273359B2 (en) 2009-09-01 2016-03-01 Yungjin Pharm. Co., Ltd. Extracellular vesicles derived from Gram-positive bacteria, and use thereof

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
BRAMLEY ET AL.: "Roles of alpha toxin and beta toxin in virulence of Staphylococcus aureus for the mouse mammary gland", INFECTION AND IMMUNITY, vol. 57, no. 8, 1989, pages 2489 - 2494 *
COLEMAN D. ET AL.: "Insertional inactivation of the Staphylococcus aureus beta toxin by bacteriophague phi-13 occurs by site and orientation-specific integration of the phi-13 genome", MOLECULAR MICROBIOLOGY, vol. 5, no. 4, 1991, pages 933 - 940 *
ODIERNO L. ET AL.: "Pathogenicity in mice of Staphylococcus aureus mutants deficient in exoprotein synthesys", VETERINARY MICROBIOLOGY, vol. 41, no. 3, August 1994 (1994-08-01), pages 249 - 258, XP023914263, DOI: doi:10.1016/0378-1135(94)90105-8 *
SANZ ET AL.: "Catalase deficiency in Staphylococcus aureus subsp. anaerobius is associated with natural loss-of-function mutations within the structural gene", MICROBIOLOGY, vol. 146, no. 2, February 2000 (2000-02-01), pages 465 - 475 *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US9273359B2 (en) 2009-09-01 2016-03-01 Yungjin Pharm. Co., Ltd. Extracellular vesicles derived from Gram-positive bacteria, and use thereof
EP2484752B1 (fr) * 2009-09-04 2016-09-21 Yungjin Pharmaceutical Co., Ltd. Vésicules extracellulaires issues de bactéries à gram positif et leur utilisation

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ES2307351A1 (es) 2008-11-16
ES2307351B2 (es) 2009-06-17

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